American Society for Biochemistry and Molecular BiologyActa Medica Okayama0021-925828052005Regulation of Chicken ccn2 Gene by Interaction between RNA cis-Element and Putative trans-Factor during Differentiation of Chondrocytes31663177ENYoshikiMukudaiBiodental Research Center, Okayama University Dental School CCN2/CTGF is a multifunctional growth factor. Our previous studies have revealed that CCN2 plays important roles in both growth and differentiation of chondrocytes and that the 3′-untranslated region (3′-UTR) of ccn2 mRNA contains a cis-repressive element of gene expression. In the present study, we found that the stability of chicken ccn2 mRNA is regulated in a differentiation stage-dependent manner in chondrocytes. We also found that stimulation by bone morphogenetic protein 2, platelet-derived growth factor, and CCN2 stabilized ccn2 mRNA in proliferating chondrocytes but that it destabilized the mRNA in prehypertrophic-hypertrophic chondrocytes. The results of a reporter gene assay revealed that the minimal repressive cis-element of the 3′-UTR of chicken ccn2 mRNA was located within the area between 100 and 150 bases from the polyadenylation tail. Moreover, the stability of ccn2 mRNA was correlated with the interaction between this cis-element and a putative 40-kDa trans-factor in nuclei and cytoplasm. In fact, the binding between them was prominent in proliferating chondrocytes and attenuated in (pre)hypertrophic chondrocytes. Stimulation by the growth factors repressed the binding in proliferating chondrocytes; however, it enhanced it in (pre)hypertrophic chondrocytes. Therefore, gene expression of ccn2 mRNA during endochondral ossification is properly regulated, at least in part, by changing the stability of the mRNA, which arises from the interaction between the RNA cis-element and putative trans-factor.No potential conflict of interest relevant to this article was reported.American Society for Biochemistry and Molecular BiologyActa Medica Okayama0021-9258295142020In vivo crystals reveal critical features of the interaction between cystic fibrosis transmembrane conductance regulator (CFTR) and the PDZ2 domain of Na+/H+ exchange cofactor NHERF144644476ENEleanor R.MartinSchool of Biological Sciences, Faculty of Biology Medicine and Health, Michael Smith Building, The University of ManchesterAlessandroBarbieriSchool of Biological Sciences, Faculty of Biology Medicine and Health, Michael Smith Building, The University of ManchesterRobert C.FordSchool of Biological Sciences, Faculty of Biology Medicine and Health, Michael Smith Building, The University of ManchesterRobert C.RobinsonResearch Institute for Interdisciplinary Science, Okayama UniversityCrystallization of recombinant proteins has been fundamental to our understanding of protein function, dysfunction, and molecular recognition. However, this information has often been gleaned under extremely nonphysiological protein, salt, and H+ concentrations. Here, we describe the development of a robust Inka1-Box (iBox)–PAK4cat system that spontaneously crystallizes in several mammalian cell types. The semi-quantitative assay described here allows the measurement of in vivo protein-protein interactions using a novel GFP-linked reporter system that produces fluorescent readouts from protein crystals. We combined this assay with in vitro X-ray crystallography and molecular dynamics studies to characterize the molecular determinants of the interaction between the PDZ2 domain of Na+/H+ exchange regulatory cofactor NHE-RF1 (NHERF1) and cystic fibrosis transmembrane conductance regulator (CFTR), a protein complex pertinent to the genetic disease cystic fibrosis. These experiments revealed the crystal structure of the extended PDZ domain of NHERF1 and indicated, contrary to what has been previously reported, that residue selection at positions −1 and −3 of the PDZ-binding motif influences the affinity and specificity of the NHERF1 PDZ2-CFTR interaction. Our results suggest that this system could be utilized to screen additional protein-protein interactions, provided they can be accommodated within the spacious iBox-PAK4cat lattice.No potential conflict of interest relevant to this article was reported.American Society for Biochemistry and Molecular BiologyActa Medica Okayama0021-925829292017Vesicular Polyamine Transporter Mediates Vesicular Storage and Release of Polyamine from Mast Cells39093918ENTomoyaTakeuchiDepartment of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesYuikaHaradaDepartment of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesSatomiMoriyamaDepartment of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKazuyukiFuruta Department of Immunobiology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesSatoshiTanaka Department of Immunobiology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesAdvanced Science Research Center, Okayama UniversityHiroshiOmoteDepartment of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesYoshinoriMoriyamaDepartment of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesMikiHiasaDepartment of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8530, hiasa@okayama-u.ac.jp. Mast cells are secretory cells that play an important role in host defense by discharging various intragranular contents, such as histamine and serotonin, upon stimulation of Fc receptors. The granules also contain spermine and spermidine, which can act as modulators of mast cell function, although the mechanism underlying vesicular storage remains unknown. Vesicular polyamine transporter (VPAT), the fourth member of the SLC18 transporter family, is an active transporter responsible for vesicular storage of spermine and spermidine in neurons. In the present study, we investigated whether VPAT functions in mast cells. RT-PCR and Western blotting indicated VPAT expression in murine bone marrow-derived mast cells (BMMCs). Immunohistochemical analysis indicated that VPAT is colocalized with VAMP3 but not with histamine, serotonin, cathepsin D, VAMP2, or VAMP7. Membrane vesicles from BMMCs accumulated spermidine upon the addition of ATP in a reserpine- and bafilomycin A1-sensitive manner. BMMCs secreted spermine and spermidine upon the addition of either antigen or A23187 in the presence of Ca2+, and the antigen-mediated release, which was shown to be temperature-dependent and sensitive to bafilomycin A1 and tetanus toxin, was significantly suppressed by VPAT gene RNA interference. Under these conditions, expression of vesicular monoamine transporter 2 was unaffected, but antigen-dependent histamine release was significantly suppressed, which was recovered by the addition of 1 mm spermine. These results strongly suggest that VPAT is expressed and is responsible for vesicular storage of spermine and spermidine in novel secretory granules that differ from histamine- and serotonin-containing granules and is involved in vesicular release of these polyamines from mast cells.No potential conflict of interest relevant to this article was reported.American Society for Biochemistry and Molecular BiologyActa Medica Okayama0021-9258292232017Crystal structure and redox properties of a novel cyanobacterial heme protein with a His/Cys heme axial ligation and a Per-Arnt-Sim (PAS)-like domain95999612ENTaikiMotomurathe Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama UniversityMichihiroSugathe Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama UniversityRainerHienerwadel the Laboratoire de Génétique et Biophysique des Plantes, UMR 7265, CNRS-CEA-Aix-Marseille Université, Faculté des Sciences de LuminyAkikoNakagawa the Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama UniversityThanh-LanLai iBiTec-SWolfgangNitschke the Laboratoire de Bioénergétique et Ingénierie des ProtéinesTakahiroKuma the Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama UniversityMiwaSugiurathe Proteo-Science Research Center, Ehime UniversityAlainBoussaciBiTec-SJian-RenShen the Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama University Photosystem II catalyzes light-induced water oxidation leading to the generation of dioxygen indispensable for sustaining aerobic life on Earth. The Photosystem II reaction center is composed of D1 and D2 proteins encoded by psbA and psbD genes, respectively. In cyanobacteria, different psbA genes are present in the genome. The thermophilic cyanobacterium Thermosynechococcus elongatus contains three psbA genes: psbA1, psbA2, and psbA3, and a new c-type heme protein, Tll0287, was found to be expressed in a strain expressing the psbA2 gene only, but the structure and function of Tll0287 are unknown. Here we solved the crystal structure of Tll0287 at a 2.0 Å resolution. The overall structure of Tll0287 was found to be similar to some kinases and sensor proteins with a Per-Arnt-Sim-like domain rather than to other c-type cytochromes. The fifth and sixth axial ligands for the heme were Cys and His, instead of the His/Met or His/His ligand pairs observed for most of the c-type hemes. The redox potential, E½, of Tll0287 was -255 ± 20 mV versus normal hydrogen electrode at pH values above 7.5. Below this pH value, the E½ increased by ≈57 mV/pH unit at 15 °C, suggesting the involvement of a protonatable group with a pKred = 7.2 ± 0.3. Possible functions of Tll0287 as a redox sensor under microaerobic conditions or a cytochrome subunit of an H2S-oxidizing system are discussed in view of the environmental conditions in which psbA2 is expressed, as well as phylogenetic analysis, structural, and sequence homologies.No potential conflict of interest relevant to this article was reported.